© 2020 The Japan Mendel Society Cytologia 85(4): 295–299

Differential Amphiplasty and Nucleolar Dominance in Somatic Metaphase Cells as Evidence of Hybridization in

Prosopis juliflora (Leguminosae, Mimosoideae)Fernando Tapia-Pastrana*

Division of Postgraduate Studies and Research, Faculty of Higher Studies Zaragoza, UNAM, México

Received July 6, 2020; accepted July 20, 2020

Summary The mitotic chromosomal complement of Prosopis juliflora from a population of Mexico was ana-lyzed using the method of air drying and Giemsa staining. In addition to an unequivocal and constant chromo-some number 2n=4x=56, detailed characteristics of its general chromosomal morphology and karyotype are described for the first time, where metacentric (m) and submetacentric (sm) chromosomes and few subtelocentric (st) predominate. Only two SAT chromosomes were recorded with microsatellites on short arms in apparently st chromosomes and their nucleolar organizer region (NOR) condition was verified by locating them close to or embedded in the nucleolus of prometaphase cells. A maximum number of two nucleoli in interphase nucleus and rarely traces of two additional micronucleoli were also corroborated. The average chromosome size exhibits a notable reduction compared to that recorded in diploid species of the genus. Taken together, these results show P. juliflora as an allotetraploid taxon whose complements show amphiplasty or nucleolar dominance. Finally, it is proposed that the current distribution of this species, adjusted mainly to coastal environments, responds to an adaptive and functional novelty as a result of a hybrid condition.

Keywords Allotetraploid, Karyotype, Microsatellite, Nucleolar organizer region, Nucleolus, SAT chromosome.

The genus Prosopis L. emend. Burkart is a genus of the Leguminosae family, traditionally included in the Mimosoideae subfamily, but currently circumscribed in the Mimosoideae clade of the Caesalpinioideae subfam-ily (Azani et al. 2017). Forty-five species of trees and shrubs have been described, of which 41 are American, three Asiatic, and one African (Burkart 1976, Schinini 1981). The genus probably originated in tropical Africa, where at present only the unspecialized, mesic species P. africana occurs (Burkart 1976). The species are widely distributed in arid and semi-arid areas where they are used as multipurpose plants as they provide food, fod-der, wood, fuel and are used as soil improvers and in environmental management (Sherry et al. 2011).

However, P. juliflora is a species that grows naturally in deciduous tropical forests and is considered a truly tropical plant, although it also develops in areas of low rainfall and a wide variety of soils (Rzedowski 1988, Trenchard et al. 2008). Originally from Mexico, its distribution has extended to some arid and semi-arid re-gions of Central and South America, Caribbean, and has been introduced in Africa and Asia (Rzedowski 1988). P. juliflora belongs to the Algarobia section, which in-cludes American species of variable habitat and greater economic importance (Burkart 1976). Species of this

section are mostly self-incompatible and cross by pol-lination insect-mediated although a small percentage of selfing reported (Solbrig and Cantino 1975, Simpson 1977, Bessega et al. 2000). The possibility of interspe-cific hybridization between species sympatric in this section has been reported (Naranjo et al. 1984, Hunziker et al. 1986, Rzedowski 1988, Galindo Almanza et al. 1992, Landeras et al. 2006, Palacios 2006).

In Mexico, P. juliflora is a shrub or small tree up to 12 m high. It extends in a vast distribution area mostly adjusted to coastal environments on the Pacific slope to Panama, Colombia, and Venezuela (Rzedowski 1988) however the status of P. juliflora in Mexico is uncertain due taxonomic confusion and misidentification (Lan-deras et al. 2006).

Prosopis is a genus characterized by a basic number x=14 and a diploid chromosome number 2n=28 exhib-ited by all cytogenetically analyzed species, with the exception of P. juliflora, the only known natural species of the genus with a 2n=4x=56 (Hunziker et al. 1975, Bukhari 1997), so the level of ploidy helps to distinguish with certainty this taxon (Harris et al. 2003, Landeras et al. 2006, Trenchard et al. 2008). Although P. juliflora is recognized as a tetraploid species, there is uncertainty about its auto or allopolyploid origin (Sherry et al. 2011).

On the other hand, it is known that plants of allopoly-ploid origin undergo the inactivation of the NORs of one of the parental genomes (Navashin 1934). NORs contain

* Corresponding author, e-mail: pasfer@unam.mxDOI: 10.1508/cytologia.85.295

296 F. Tapia-Pastrana Cytologia 85(4)

tandemly arranged highly reiterated rRNA genes cod-ing for 18S-5.8S-26S rRNA whose expression is under epigenetic control (Pikaard 2000). These genes are generally associated with a secondary constrictions and satellites on so-called SAT chromosomes. Interspecific hybrids often have rRNA genes of one parent function-ally dominant over the rRNA genes of the other parent. This epigenetic phenomenon is known as nucleolar dom-inance (Navashin 1934, Wallace and Langridge 1971) and there are many examples of such regulation of rRNA gene activity in allopolyploids (Chen and Pikaard 1997a, b, Pikaard 2000, Pires et al. 2004). In other words, the secondary constriction of the SAT chromosome of one of the parental species is missing in the hybrid and the satellite is retracted onto the chromosome arm as a con-sequence (Lacadena and Cermeño 1985).

In autopolyploids, for example, the number of satel-lites present in a diploid species is also doubled, since this does not imply loss or suppression of nucleolar function, the NOR regions associated with secondary constrictions on SAT chromosomes are lax and there-fore, the satellites appear clearly. Plants as Medicago sa-tiva, a recognized autotetraploid exhibit four satellites in metaphase cells (Falistocco 1987). In contrast, plants of allopolyploid origin as cotton (Gossypium hirsutum, En-drizzi et al. 1985), wheat (Triticum aestivum, Lacadena and Cermeño 1985), and canola (Brassica napus, Xiong and Pires 2011), undergo inactivation of the NOR of one of the parental genomes, silenced by the effect of nucleo-lar dominance and consequently a smaller number of satellites is recorded (Doyle et al. 2008, Ge et al. 2013). The NOR competition is cytologically expressed as amphiplasty: a term proposed to denote morphological changes that occur in chromosomes following interspe-cific hybridization (Navashin 1934, Rieger et al. 1976).

The objective of this work is (1) to verify by chromo-somal count the ploidy level of individuals of P. juliflora from the Mexican population, (2) corroborate the NOR condition of the SAT chromosomes (3) use the nucleolar

dominance criterion to identify the auto or allopolyploid origin of P. juliflora.

Materials and methods

P. juliflora seeds were collected during the spring of 2015 from three various trees in Playa Careyi-tos, Municipality of La Huerta, Jalisco State, Mexico (19.437396, -105.026201). The voucher specimens were deposited in the National Herbarium (MEXU) of the In-stituto de Biología, UNAM.

Batches of 15 healthy seeds from each of the three trees were used. After being mechanically scarified, they were placed in Petri dishes lined with wet filter paper and allowed to germinate at room temperature and in natural light.

Interphase nuclei and chromosomes at metaphase and prometaphase were obtained following the splash meth-od (Tapia-Pastrana and Mercado-Ruaro 2001). A least 15 root meristems were collected from 5.0–8.0 mm long roots pretreated with 2 mM 8-hydroxyquinolin for 5 h at room temperature and fixed in the fixative (ethanol : ace-tic acid=3 : 1). They were then treated with a mixture of 20% pectinase (Sigma) and 2% cellulase (Sigma) in 75 mM KCl for 80 min at 37°C. After centrifugation at 1,500 rpm for 10 min, the cell pellet was transferred to 75 mM KCl solution for 17 min at 37°C. After two suc-cessive rinses with the KCl solution, they were again fixed in the fixative and subsequently rinsed twice more. One or two drops of the suspension of pellets were placed on clean slides, air-dried, and stained in 10% Giemsa for 13 min. Preparations were made permanent using a synthetic resin.

At least ten metaphase plates of three plants with well-spread chromosomes, no chromosome overlapping, and the same contraction and five prophase plates were photographed from each collection, using a microscope (Axioscope, Carl Zeiss) and analyzed for chromosome number determinations. Seven photographs of meta-

Fig. 1. Chromosomes of P. juliflora of plants from population of Playa Careyitos, Jal. A: Metaphase chromosome plate showing a 2n=4x=56 and chromosomes with the optimal distribution. Arrows point to a pair of SAT chromosomes, in this case, st, with lax secondary constrictions associated with satellites. B: Prometaphase nucleus 2n=4x=56. Arrows point to two embedded satellites in a single nucleolus (N), showing that SAT chromosomes carry NORs. Scale bars=10 µm.

2020 Evidence of Hybridization in Prosopis juliflora 297

phases with chromosomes having similar comparable degrees of contraction were utilized to obtain the total chromosome length (TCL), mean chromosome length (MC), and the difference in length between the longest chromosome and the shortest chromosome (Range).

Results

In total, 1,110 metaphase plates were observed, of which 1,101 (98.18%) exhibited 2n=4x=56 and only nine nuclei showed 2n˃56. The average chromosome size was 1.01±0.06 µm. The precise location of the centromere was only possible on the largest chromo-somes and almost impossible on the smallest, which prevented the proper description of a karyotype for-mula. The chromosome complement is dominated by m and sm chromosomes with few st chromosomes. TCL was=57.1±3.6 µm and MC=1.01±0.06 µm. Chromo-somal size ranges from 1.43 to 0.76 µm. The metaphase plates displayed a pair of SAT chromosomes, apparently st, with secondary constrictions and microsatellites in the short arms (Fig. 1A). These were frequently ob-served close to or even embedded in the nucleoli of cells at prometaphase, confirming that they carry the NOR (Fig. 1B).

On the other hand, the interphase nuclei constantly showed the presence of two strongly stained nucleoli; one slightly larger than the other (Fig. 2A), and occa-sionally two additional micronucleoli were also recorded on the three plants (Fig. 2B).

Discussion

Species belonging to the Algarobia section of Proso-pis are characterized by having predominantly haploid chromosome numbers n=14 and diploid 2n=2x=28 according to the record of the Plant Chromosome Num-bers Index (http://www.tropicos.org/Project/IPCN) and it is believed that reports of polyploidy in them may represent polysomaty in root tissues (Cherubini 1981, Trenchard et al. 2008). On the other hand, flow cytom-

etry has recently indicated that P. juliflora is completely tetraploid with a somatic chromosome number of 2n=56 (Trenchard et al. 2008) and interestingly, in the same country this species presents vast areas of distribution mostly associated to coastal environments where pre-cipitation annual average reaches 1,500 mm (Rzedowski 1988).

Although there is agreement about the basic number and the diploid condition of most species of the genus Prosopis, few cytogenetic studies have been able to es-tablish karyotype formulas due mainly to the small size of the chromosomes, which ranges from 0.5 to 1.3 µm (Hunziker et al. 1986). However, in populations of P. laevigata (Tapia Pastrana et al. 1999, Tapia-Pastrana and Mercado-Ruaro 2001), karyotype formulas were deter-mined and two SAT chromosomes were identified. On this basis, a similar condition could be expected for the remaining species in Algarobia.

The results obtained here confirm that in P. juliflora the SAT chromosomes also carry NOR. Likewise, the number of secondary constrictions shows an exact corre-spondence with the maximum number of nucleoli (two) observed in the root tip interphase cells, as expected in hybrid individuals that experience nucleolar dominance (Wilkinson 1944, Keep 1960, 1962, Wallace and Lan-gridge 1971). The presence of extra micronucleoli is a surprising result and it can be speculated that these rep-resent traces of subdominant rRNA genes selectively not silenced in this particular tissue (Pikaard 2000). A better response to this phenomenon will be obtained when the mechanism responsible for the initial discrimination be-tween the parental sets of rRNA genes is better known (Pikaard 2000).

In the present work, the high number of somatic cells analyzed in three plants confirmed that P. juliflora was an entirely tetraploid species. On the other hand, the constant presence of a pair of SAT chromosomes re-solves the origin of the polyploidy exhibited by this spe-cies. It is an allotetraploid taxon derived from a hybrid-ization process whose parents are still unknown.

The results show that both nucleolar dominances, the frequently observed suppression of nucleolus forma-tion, and differential amphiplasty, that is, the retraction of secondary constrictions and associated satellites, are manifested in P. juliflora cells and that the polyploid condition induces changes that affect all chromosomes. The latter is manifested in a notable reduction in the size of the chromosomes since P. juliflora exhibits the lowest MC (1.01 µm) among the species of the genus in which this cytogenetic parameter has been recorded (Hunziker et al. 1986, Tapia-Pastrana and Mercado-Ruaro 2001) and this is consistent with the amphiplasty described in a series of karyological investigations in interspecific hybrids of Crepis (Navashin 1928, 1934).

Furthermore, if we consider that polyploidy in con-cert with hybridization increases genetic diversity and

Fig. 2. Interphase cells of P. juliflora. A: Nucleus exhibits two nucleoli, one slightly larger than the other. B: Nucleus exhib-its, in addition to the two frequently recorded nucleoli, two extra micronucleoli. Arrows indicate the common type of interphase nucleoli. Arrowheads point to uncommon micro-nucleoli. Nu=Nucleus. Scale bars=10 µm.

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promotes the acquisition of new functional and adaptive traits concerning diploid progenitors, then the current distribution of P. juliflora associated with coastal envi-ronments could be considered a novelty or ecological specialization. Finally, knowledge of the cytogenetic characteristics of P. juliflora should contribute to a bet-ter understanding of its genetic system, to the clarifica-tion of its center of origin and evolution, and a better interpretation of its taxonomic relationships. This goal must be achieved soon because, in the absence of a clear mechanism that explains which dominant and subdomi-nant rRNA genes are discriminated in newly formed hy-brids (Pikaard 2000), the size of the secondary constric-tions and the shape of the satellites in P. juliflora can provide clues as to which genes are expressed during the epigenetic phenomenon of nucleolar dominance.

Acknowledgements

The author is grateful for the support of the Division of Postgraduate Studies and Research, Faculty of Higher Studies Zaragoza, UNAM.

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